(Supported by the NIH/NCRR/P41RR 01219 grant) Flagella and cilia are variants of a microtubule-based organelle that is the source of motility for metazoan sperm, several species of protozoa, and metazoan epithelial cells that propel extracellular fluid across tissue surfaces. The most common structural motif for flagella consists of 9 doublet microtubules (MTs) cylindrically arranged around two singlet microtubules. Wave motion is generated in flagella by the MT motor protein dynein which is arranged in two longitudinal rows along the outer doublet MTs. The rows of outer dynein arms have a 24nm longitudinal periodicity, while the periodicity of the inner arms is still controversial. The outer doublet MTs are linked to the central pair via radial spokes that occur three per 96 nm. There is also a regular lattice of accessory components along the central pair MTs. Genetic studies have identified numerous mutant forms of flagella and simple averaging along peri odic com ponents often yields sufficient resolution to construct difference maps between wild type and deletion mutants, thereby locating specific gene products. Hitherto, structural studies of flagella have been limited by specimen preparation. Superior structural preservation and resolution is obtained with frozen-hydrated specimens. In previous work a preliminary application of cryo-EM to sperm flagella yielded 4 nm resolution in projection. Unfortunately, frozen-hydrated specimens are more labile in the electron beam, which, along with the structural complexity of flagella and the inability to align single images of different specimens, prevented computing a 3D reconstruction of flagella. Recent technical improvements have reduced the dose required to record a tilt series, enabling tomographic reconstruction of frozen-hydrated specimens. Six reconstructions of unfixed, unstained, sea urchin sperm axonemes embedded in vitreous ice have been made from so far. An initial problem with loss of microtubules was traced to dilution of the high-salt buffer, which was required to retain the integrity of the gold bead markers needed for alignment of the tilt images. This problem was overcome by partial substitution of the high-salt buffer with sucrose buffer. Experience with cryo-tomography was gained using both axonemes and mitochondria (see TRD project "Development of cryofixation techniques for mitochondria"), as the two projects were carried out in parallel by the same personnel. Improvements in imaging techniques, including reduction of contamination, selection of optimal defocus values, and decreased electron dose made possible by a new scintillator for the CCD camera, led to increased resolution. We can now reliably obtain 4nm layer lines, corresponding to the microtubule subunit repeat, in power spectra of images of suitable axonemes. The 3-D resolution of the best tomographic reconstructions is approximately 5nm. The reconstructions now show all the features expected in the axoneme: all 9+2 microtubule sets, the periodic central sheath material, the radial spokes, and the inner and outer dynein arms. The subunits of the microtubules can not quite be resolved in slices from the reconstructions. The axonemes are flattened by approximately 50%. The resolution can be improved by further experiments with the optimal ice thickness. The ice thickness is controlled by modifications of the blotting and drying techniques used prior to plunge-freezing. The flattening is also believed to be due to the blotting/drying method. Work is now underway to apply averaging techniques to the repeating structures along the length of the axoneme, which should give more detailed structural information. The work was presented at the International Congress on Electron Microscopy in Cancun in September. McEwen, B.F., Hsieh, C.-E., Marko, M., Buttle, K., Frank, J. (1998) Cryo-electron tomography applied to sperm flagella. Electron Microscopy 1998 (H. Benavides and M. Yacaman, Eds.), Institute of Physics, Bristol and Philadelphia, Vol IV, pp. 459-460.